Many different fluorescent compounds have been used to detect nucleic acids. Broadly, fluorescent labels of nucleic acids can be divided into two classes: (1) those which covalently modify nucleic acids with a fluorescent moiety, and (2) those which non-covalently modify nucleic acids with a fluorescent moiety, i.e., by ionic interactions, hydrogen-bonding, or intercalation. In general, non-covalent fluorescent probes of nucleic acids exhibit dramatically increased fluorescence upon binding to nucleic acids, and consequently, have been very useful in assays designed to determine the total nucleic acid present in a given sample. In addition, non-covalently bound fluorescent molecules can, and will, migrate from a labeled strand to an unlabeled one. Covalently bound fluorescent molecules, on the other hand, can not migrate from a labeled oligonucleotide to an unlabeled one. Therefore covalently bound fluorescent moieties are preferred for use as fluorescently tagged nucleic acid probes.
Examples of fluorescent compounds which have been covalently attached to nucleic acid sequences include conjugates between nucleotide triphosphates or phosphoramidites and fluorescent moieties, and directly reactive dyes. Nucleotide triphosphates are incorporated into a nucleic acids by nucleic acid polymerases. Commercially available nucleotide triphosphates-dye conjugates include dCTP-Cy3, dCTP-Cy5, dUTP-FluorX, etc. available from DuPont, Molecular Probes, Boehringer Mannheim, and Amersham Life Sciences. These dye conjugates contain cyanine or fluorescein derivatives which are covalently bound to the nucleotide, and each dye conjugate differs with respect to the absorbance maxima of the dye moiety. Directly reactive dyes covalently bind to an existing nucleic acid sequence. A few reactive dyes are commercially available, including various psoralens and ethidium mono- and di-azides.
The chemistry associated with conjugates of phosphoramidites and fluorescent molecules has dramatically improved in recent years allowing for the complete synthesis of fluorescently labeled oligonucleotides with commercially available nucleic acid synthesizers. Fluorescently labeled oligonucleotides have also been synthesized by a combination of modified phosphoramidites and reactive dyes, typically involving the incorporation of primary amines in the oligonucleotide during synthesis followed by covalent coupling of the amine groups to a reactive dye.
Of the three methods for the covalent linkage of fluorescent compounds to oligonucleotides, the nucleotide triphosphate-dye conjugates offer the greatest flexibility and the highest achievable specific fluorescence. Synthetic nucleic acids (molecules produced non-enzymatically) are generally limited to less than 100 bases and are subject to variable dye coupling chemistries. Directly reactive dyes, such as ethidium monoazide, react non-specifically and can potentially damage the labeled oligonucleotide. Polymerase-driven labeling, on the other hand, can produce molecules from a few tens of bases to several kilobases, can utilize standard labeling methods such as nick translation and primer extension reactions, and the degree of dye incorporation can be roughly controlled by varying the ratio of labeled NTP to unlabeled NTP.
The primary limitation of polymerase-driven fluorescent labeling of nucleic acids is the absence of absolute control of the amount of fluorescent compound incorporated into a particular sequence. For example, if one desires to label DNA with dCTP-Cy3 and the specific sequence has only a limited number of "C" sites, then the resulting fluorescently labeled oligonucleotide will have relatively few Cy3 molecules and consequently a low specific fluorescence. The present invention overcomes this sequence specific limitation and optimizes the incorporation of the fluorescent moiety by polymerase.